Use of collagenase in the preparation of neural stem cell cultures

ABSTRACT

The invention provides a method for using collagenase to dissociate neural stem cells in neural stem cell cultures. The collagenase treatment results in an increased cell viability and an increased number of proliferated neural stem cells over time.

FIELD OF THE INVENTION

[0001] This invention relates generally to a production or manufacturingprocess for the in vitro proliferation and culture of neural stem cellcultures using collagenase to dissociate the cultured cells.

BACKGROUND OF THE INVENTION

[0002] The development of the mammalian central nervous system (CNS)begins in the early stage of fetal development and continues until thepost-natal period. The mature mammalian CNS is composed of neuronalcells (neurons), and glial cells (astrocytes and oligodendrocytes). Thefirst step in neural development is cell birth, which is the precisetemporal and spatial sequence in which stem cells and stem cell progeny(i.e. daughter stem cells and progenitor cells) proliferate.

[0003] One identifying feature of a stem cell is its ability to exhibitself-renewal or to generate more of itself. A definition of a stem cellis provided by Potten & Loeffler, 110 Development 1001 (1990) who havedefined stem cells as “undifferentiated cells capable of (a)proliferation, (b) self-maintenance, (c) the production of a largenumber of differentiated functional progeny, (d) regenerating the tissueafter injury, and (e) a flexibility in the use of these options.” Therole of stem cells is to replace cells that are lost by natural celldeath, injury or disease.

[0004] U.S. Pat. Nos. 5,750,376, 5,851,832 (both naming Weiss) and U.S.Pat. No. 5,753,506 (Johe), each incorporated herein by reference, referto in vitro cultures containing neural stem cells. The Weiss patentsrefer to both suspension and adherent culture, while Johe refers toparticular adherent cultures. When the cells are propagated asneurospheres in suspension culture, within 3-4 days in the presence of aproliferation-inducing growth factor, a multipotent neural stem cellbegins to divide giving rise to a cluster of undifferentiated cellsreferred to as a “neurosphere”. The cells of a single neurosphere areclonal in nature because they are the progeny of a single neural stemcell. In the continued presence of one or more proliferation-inducinggrowth factors, such as EGF, bFGF, or the like (and combinationsthereof), cells within the neurosphere continue to divide resulting inan increase in the size of the neurosphere and the number ofundifferentiated cells. The cells within the neurosphere areimmunoreactive for nestin, an intermediate filament protein found inmany types of undifferentiated CNS cells. In contrast, maturedifferentiated cell types derived from the neural stem cell progeny arepredominantly negative for nestin.

[0005] In the prior art, the cells in the cluster were mechanicallydissociated by trituration to produce single cells between passages.Trituration, because it is a mechanical process, exerts shear forces onthe cells, that may reduce cell viability between passages. The objectof the present invention is to provide an improved culture andmanufacture process that increases cell viability from passage topassage and maintains more of the most primitive cells (with thegreatest differentiation potential and self-renewal capability).

SUMMARY OF THE INVENTION

[0006] The invention provides a method for the in vitro proliferation ofmultipotent neural stem cell culture using collagenase to dissociatecells in neurospheres between passages. According to the method of thisinvention, use of collagenase results in improved neural stem cellculture viability, increased number of proliferated cells in thosecultures over time, and improved maintenance of cell cultures, ascompared with dissociation by trituration or other enzymatic treatmentssuch as trypsinization.

BRIEF DESCRIPTION OF THE FIGURES

[0007]FIG. 1 shows a comparison between the prior art trituration methodof dissociating proliferating human neurospheres and the collagenasemethod of the invention, demonstrating an increased number of viablecells over time using the collagenase method.

DETAILED DESCRIPTION OF THE INVENTION

[0008] Introduction.

[0009] The invention provides a novel manufacturing process forproliferating neural stem cell cultures, using collagenase to dissociateneurospheres (“aggregated” cells). This method results in anunexpectedly improved neural stem cell culture viability and increasednumber of proliferated cells over time, as compared with prior arttrituration and trypsinization methods for dissociating neural stem cellcultures.

[0010] In one embodiment of the collagenase manufacture process,neurosphere cells are harvested and centrifuged (e,g., at 1000 rpm for3-5 min). After aspirating medium, neurospheres are resuspendedcollagenase solution (e.g., 1 ml of pre-warmed (37° C.) 0.5 mg/ml), andincubated with the collagenase. After the incubation, cell suspension isdiluted in medium and cultured.

[0011] In the prior art trituration method, neurosphere cells wereharvested and centrifuged. After aspirating medium, neurospheres wereresuspended in ˜200 μl of medium. Neurospheres were triturated by usinga pipette (e.g., a P200 pipetman with 75 μl volumes, about 100 times).Then, the cell suspension was diluted in medium and cultured.

[0012] Collagenase.

[0013] Any collagenase which is effective for dissociating neural stemcells in culture may be used in the manufacture process of theinvention. “Collagenase” is an enzyme that digests the extracellularmatrix protein collagen (Harper, 49 Ann. Rev. Biochem. 1063 (1980)). Onesource of collagenase is the bacterium Clostridium histolyticum. Onecollagenase assay is a modification of Mandl et al., 32 J. Clin. Invest.1323 (1953), whereby collagenase is incubated for 5 hr with nativecollagen. The extent of collagen breakdown is determined using thecolorimetric ninhydrin method of Moore & Stein, 176 Biol. Chem 367(1948). For collagenase unit definition, 1 unit releases one μmole ofL-leucine equivalents from collagen in 5 hr at 37° C., pH 7.5.

[0014] Crude collagenases can be used for cell dissociation procedures.Crude collagenase preparations contain not only several collagenases butalso a sulfhydryl protease, clostripain, a trypsin-like enzyme, and anaminopeptidase. In some embodiments, we prefer crude collagenasepreparations, because of the presence of these additional activities.This combination of collagenolytic and proteolytic activities iseffective at breaking down intercellular matrices, the essential part oftissue dissociation. Crude collagenase is inhibited by metal chelatingagents such as cysteine, EDTA or o-phenanthroline. It is also inhibitedby alpha-2-macroglobulin, a large plasma glycoprotein. Ca²⁺ is thoughtto be required for enzyme activity.

[0015] Commercially-available sources of collagenase are useful in themethods of this invention. For example, purified collagenase containsminimal secondary proteolytic activities, but with high collagenaseactivity. Purified collagenase can be collagenase H (Cat # 1 087 789)from Boerhinger Mannheim (Indianapolis, Ind.). A stock solution of 0.5mg/ml collagenase is prepared in DPBS containing 0.1% BSA, and stored−20 C. Other commercially available sources are Dispase (BoehringerMannheim), Liberase (Boehringer Mannheim) or collagenase (Serva). Therange of collagenase used can be from 100-1000 μg/ml (18-180 mU/ml),preferably between 300-700 μg/ml, (54-126 mU/ml) most preferably about500 μg/ml (90 mU/ml).

[0016] Isolation and in vitro Proliferation of Multipotent Self-renewingCNS Neural Stem Cells

[0017] Neurobiologists have used various terms interchangeably todescribe the undifferentiated cells of the CNS. Terms such as “stemcell”, “precursor cell”, and progenitor cell” were once used in thescientific literature. However, there are different types ofundifferentiated neural cells, with differing characteristics and fates.The terminology used for undifferentiated multipotent neural cells hasevolved such that these cells are now termed “neural stem cells” U.S.Pat. No. 5,750,376 defines the “neural stem” cell proliferated in vitroto mean “an oligopotent or multipotent stem cell which is able to dividewithout limit and under specific conditions can produce daughter cellswhich terminally differentiate into neurons and glia.” The capability ofa cell to divide without limit and produce daughter cells whichterminally differentiate into neurons and glia are CNS stem cellcharacteristics. A CNS neural stem cell is capable of self maintenance,meaning that with each cell division, one daughter cell will also be astem cell. A CNS neural stem cell can be induced to proliferate usingthe methods of the present invention.

[0018] The non-stem cell progeny of a neural stem cell may includeprogenitor cells. The progenitor cells generated from a singlemultipotent self-renewing CNS neural stem cell are capable ofdifferentiating into neurons, astrocytes (type I and type II) oroligodendrocytes. By contrast, the CNS neural stem cell is “multipotent”because its progeny have multiple differentiative pathways.

[0019] A “neural progenitor cell” is an undifferentiated cell derivedfrom a multipotent self-renewing CNS neural stem cell, and is not itselfa stem cell. Some progenitor cells can produce progeny that are capableof differentiating into more than one cell type. For example, an O-2Acell is a glial progenitor cell that gives rise to oligodendrocytes andtype II astrocytes, and thus could be termed a “bipotential” progenitorcell. A distinguishing feature of a progenitor cell is that, unlike astem cell, it has limited proliferative ability and thus does notexhibit self maintenance. It is committed to a particular path ofdifferentiation and will, under appropriate conditions, eventuallydifferentiate into glia or neurons.

[0020] The term “precursor cells” refers to the progeny of multipotentself-renewing CNS neural stem cells, and thus includes both progenitorcells and daughter multipotent self-renewing CNS neural stem cells.

[0021] Multipotent self-renewing CNS neural stem cells can be obtainedfrom embryonic, post-natal, juvenile or adult neural tissue. Thepreferred source neural tissue is from mammals, preferably rodents(e.g., mice and rats) and primates, and most preferably, from humans.Method for the isolation, proliferation, and passaging of multipotentself-renewing CNS neural stem cells from adult human neural tissue,embryonic human neural tissue, adult monkey (Rhesus) neural tissue,mouse embryonic neural tissue, and juvenile and adult mouse braintissue, including the establishment of neural stem cells in culture fromCNS neural stem cells as well as the differentiation of the CNS neuralstem cell progeny, are provided by Weiss et al., U.S. Pat. Nos.5,750,376 and 5,851,832 (each incorporated herein by reference). In themethod of the present invention however, neurospheres arecollagenase-treated to dissociate the aggregated cells, rather thantriturated or trypsinized according to the methods used by Weiss et al.,U.S. Pat. Nos. 5,750,376 and 5,851,832 and Johe, U.S. Pat. No. 5,753,506(each incorporated herein by reference).

[0022] Multipotent self-renewing CNS neural stem cells can be obtainedfrom donor tissue by dissociation of individual cells from theconnecting extracellular matrix of the tissue, as described by Weiss etal, U.S. Pat. Nos. 5,750,376 and 5,851,832 and Johe, U.S. Pat. No.5,753,506. Tissue is removed from a neural region using a sterileprocedure, and the cells are dissociated in tissue culture medium usingany method known in the art including treatment with enzymes such astrypsin, collagenase and the like, or by using physical methods ofdissociation such as with a blunt instrument, as described by Weiss etal, U.S. Pat. Nos. 5,750,376 and 5,851,832. Dissociated cells arecentrifuged at low speed, between 200 and 2000 rpm, usually between 400and 1000 rpm, and then resuspended in culture medium. The neural cellscan be cultured in suspension or on a fixed substrate. Cell suspensionsare seeded in any receptacle capable of sustaining cells, particularlyculture flasks, culture plates or roller bottles, and more particularlyin small culture flasks such as 25 cm² culture flasks. Cells cultured insuspension are resuspended at approximately 5×10⁴ to 1×10⁶ cells/ml,preferably 1×10⁶ cells/ml (for 20 week g.w. tissue). Cells plated on afixed substrate are plated at approximately 2-3×10³ 10 cells/cm²,preferably 2.5×10³ cells/cm².

[0023] Collagenase-treated neural stem cell cultures, including themultipotent self-renewing CNS neural stem cells of the neurospheres, canbe proliferated either on substrates or in suspension, preferablyforming clusters of associated undifferentiated cells, referred to as“neurospheres.” After culture in the absence of a substrate, theproliferating neurospheres lift off the floor of the culture dish andtend to form the free-floating clusters characteristic of neurospheres.The proliferating precursor cells of the neurosphere continue toproliferate in suspension. The neurospheres of the suspension culturecan be easily passaged to reinitiate proliferation. In the method of theinvention, individual cells in the neurospheres are separated bycollagenase treatment. The collagenase-treated neurosphere cells arethen replated at the desired density to reinitiate proliferation. Singlecells from the dissociated neurospheres are suspended in culture mediumcontaining growth factor, and a percentage of these cells proliferateand form new neurospheres largely composed of undifferentiated cells.This manufacture process can be repeated to result in a logarithmicincrease in the number of viable cells at each passage. The procedure iscontinued until the desired number of cells is obtained.

[0024] Weiss et al., U.S. Pat. Nos. 5,750,376 and 5,851,832 disclose“culture medium containing one or more predetermined growth factorseffective for inducing multipotent neural stem cell proliferation.”However, different basal media can be used, including, but not limitedto:

[0025] D-MEM/F12 (Gibco BRL, Gaithersburg, Md.);

[0026] Ex Vivo 15 (Bio Whittaker, Walkersville, Md.);

[0027] Neural progenitor basal media, (Clonetics. San Diego, Calif.); or

[0028] combination of the basal media listed above.

[0029] The culture medium is supplemented with at least oneproliferation-inducing growth factor.

[0030] As used herein, the term “growth factor” refers to a protein,peptide or other molecule having a growth, proliferative,differentiative, or trophic effect on neural stem cells and/or neuralstem cell progeny. Growth factors which may be used for inducingproliferation include any trophic factor that allows neural stem cellsand precursor cells to proliferate, including any molecule which bindsto a receptor on the surface of the cell to exert a trophic, orgrowth-inducing effect on the cell. Preferred proliferation-inducinggrowth factors include members of the EGF superfamily, FGF superfamily,and TGF-α superfamily, such as EGF, amphiregulin, acidic fibroblastgrowth factor (aFGF or FGF-1), basic fibroblast growth factor (bFGF orFGF-2), transforming growth factor alpha (TGF-α), leukocyte inhibitoryfactor (LIF), glycostatin C and combinations thereof A preferredcombination of proliferation-inducing growth factors is EGF or TGF-Awith FGF-1 or FGF-2. Growth factors are usually added to the culturemedium at concentrations ranging between about 1 fg/ml to 1 mg/ml.Concentrations between about 1 to 100 ng/ml are usually sufficient.Simple titration experiments can easily be performed to determine theoptimal concentration of a particular growth factor.

[0031] The optimization of media formulation permits a higher percentageof neurospheres initiated from primary brain tissue to be established.We prefer Ex Vivo 15 media. The optimization of media formulation alsopermits a more consistent growth of neurospheres. To maximizeneurosphere development, the collagenase-treated neurosphere cells aretypically cultured in the presence of LIF, bFGF, EGF, and neuralsurvival factor, NSF (Cat. CC-4323, Clonetics, San Diego, Calif.).

[0032] A typical media formulation to culture human neural stem cellcultures is provided in TABLE 1. TABLE 1 Serum-Free N2/EGF SupplementedCulture Medium For Neural Stem Cell Cultures Quantity Reagents 87 mlDMEM/F12 (Gibco lot. 1012915; Cat. No. 11330-032) 1 ml N-2 Supplement(Gibco lot 1017018; Cat. No. 17502-014) 1 ml 0.2 mg/ml heparin (Sigmalot 28H0320; Cat. No. H-3149) 1 ml 0.2M Glutamine (JCR lot 7N2320; Cat.No. 59202-77p) 10 ml 3% Glucose (Sigma, lot 37H0841; Cat. No. G-7021) 20μl 100 μg/ml EGF (R&D lot CE107091; Cat. No. 236-EG) 100 μl 20 μg/mlFGF-2 (Gibco lot KCQ411; Cat. No. 13256-029) 100 μl 10 μg/ml LIF (R&Dlot OX038021; Cat. No. 250-L)

[0033] EGF is added to 100 ml base medium for human neural stem cellcultures after filtering the medium. EGF is relatively stable in themedium. FGF-2 and LIF are added when medium is ready to use. The finalconcentrations of the supplement reagents are: TABLE 2 5 μg/ml Insulin100 μg/ml Human transferrin 6.3 ng/ml Progesterone 16.1 μg/ml Putrascine5.2 ng/ml Selenite 20 ng/ml EGF 20 ng/ml FGF-2 10 ng/ml LIF 2 μg/mlheparin 2 mM L-glumtamine 6 mg/ml Glucose

[0034] Collagenase-treated neural stem cell cultures can also bedifferentiated using the differentiation paradigms as described in Weisset al., U.S. Pat. Nos. 5,750,376 and 5,851,832. For example, (1)collagenase-treated neural stem cell cultures can be differentiated by arapid differentiation after being plated on poly-L-ornithine-coatedglass coverslips in medium containing 0.5% fetal bovine serum (FBS);.(2) collagenase-treated neural stem cell cultures can be differentiatedusing dissociated neurospheres in EGF-free complete medium containing 1%FBS; (3) collagenase-treated neural stem cell cultures can bedifferentiated using single neurospheres plated onto laminin-coatedglass coverslips; (4) collagenase-treated neural stem cell cultures canbe differentiated using single dissociated neurospheres,collagenase-treated, and plated onto a 35 mm culture dish; (5)collagenase-treated neural stem cell cultures can be differentiatedusing neurospheres co-cultured with striatal astrocytes. In a preferredmethod of differentiation, neurosphere cells are plated on a laminincoated substrate in the presence of FBS. The resulting differentiatedcells are probed by indirect immunocytochemistry for the presence ofneuron, astrocytes and oligodendrocytes, for example, using antibodiesto MAP-2, tau-1, neurofilament 168 kDa, β-tubulin, GABA, substance P(neuronal markers), GFAP (astrocytic marker), O4, and MBP(oligodendrocyte markers). All three neural cell types are expected tobe identified.

[0035] Genetic Modification of Collagenase-treated Neural Stem CellCultures.

[0036] The neural stem cell cultures described herein may be geneticallymodified according to any suitable method known in the art, including invitro genetic modification, or generation of genetically modified neuralstem cell cultures form transgenic mammals. The genetic modification ofneural stem cells is performed either by infection with recombinantretroviruses or transfection using methods known in the art (see,Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory, N.Y., 1989). Methods of making genetically modifiedneural stem cells cultures are described, e.g., Weiss, U.S. Pat. No.5,750,376, incorporated herein by reference.

[0037] Generally, the term “genetic modification” refers to the stableor transient alteration of the genotype of a precursor cell byintentional introduction of exogenous DNA. DNA may be synthetic, ornaturally derived, and may contain genes, portions of genes, or otheruseful DNA sequences. The term “genetic modification” includes variousgene activation methods known in the art. See, e.g., U.S. Pat. Nos.5,733,761 and 5,733,746, each herein incorporated by reference.

[0038] In certain embodiments the neural stem cells are geneticallymodified to produce a biologically active molecule, including hormones,enzymes, neurotransmitters, antibodies, cytokines, lymphokines, growthfactors, trophic factors, or biological response modifiers.Alternatively the neural stem cells are genetically modified to providea metabolic or immunologic function upon implantation in a host,preferably a human. It may also be desired to genetically modify cellsso they secrete a certain growth factor product. The term “growth factorproduct” refers to a protein, peptide, mitogen, or other molecule havinga growth, proliferative, differentiative, or trophic effect (e.g., NGF,BDNF, the neurotrophins, CNTF, amphiregulin, FGF-1, FGF-2, EGF, TGF-α,TGF-βs, PDGF, IGFs, and the interleukins). Neurosphere progeny cells canalso be modified to express a certain growth factor receptor (e.g., p75low affinity NGF receptor, CNTF receptor, the irk family of neurotrophinreceptors, EGF-R, FGF-R, and amphiregulin receptors).Collagenase-treated neural stem cell cultures can be engineered toproduce various neurotransmitters, neurotransmitter receptors, orneurotransmitter-synthesizing enzymes.

[0039] Transplantation of Neural Stem Cell Cultures to Alleviate HumanDisorders.

[0040] Collagenase-treated neural stem cell cultures can be produced andtransplanted into mammalian hosts, preferably human patients, for thetreatment of various disorders, both in the central nervous system(“CNS”) and systemically. Cells are delivered to the subject by anysuitable means known in the art. If delivered to the central nervoussystem, then the cells are administered to a particular region using anymethod which maintains the integrity of surrounding areas of the brain,preferably by injection cannula. Injection methods exemplified by thoseused by Duncan et al., 17 J. Neurocytology 351-361 (1988), and scaled upand modified for use in humans are preferred. Methods for the injectionof cell suspensions such as fibroblasts into the CNS may also beemployed for injection of neural precursor cells. Additional approachesand methods may be found in Neural Grafting in the Mammalian CNS,Bjorklund & Stenevi, eds. (1985).

[0041] Collagenase-treated neural stem cell cultures can be produced andtransplanted using the above procedures to treat variousneurodegenerative disorders. Such CNS disorders encompass numerousafflictions such as neurodegenerative diseases (e.g. Alzheimer's andParkinson's), acute brain injury (e.g. stroke, head injury, cerebralpalsy) and a large number of CNS dysfunctions (e.g. depression,epilepsy, and schizophrenia). In recent years neurodegenerative diseasehas become an important concern due to the expanding elderly populationwhich is at greatest risk for these disorders. These diseases, whichinclude Alzheimer's Disease, Multiple Sclerosis (MS), Huntington'sDisease, Amyotrophic Lateral Sclerosis, and Parkinson's Disease, havebeen linked to the degeneration of neural cells in particular locationsof the CNS, leading to the inability of these cells or the brain regionto carry out their intended function. By providing for maturation,proliferation and differentiation into one or more selected lineagesthrough specific different growth factors the progenitor cells may beused as a source of committed cells. In one series of embodiments,collagenase-treated neural stem cell cultures can be produced andtransplanted using the above procedures for the treatment ofdemyelination diseases. Any suitable method for the implantation ofcells near to the demyelinated targets may be used so that the cells canbecome associated with the demyelinated axons.

[0042] Neural stem cell cultures made according to the present inventionmay also be used to produce a variety of blood cell types, includingmyeloid and lymphoid cells, as well as early hematopoietic cells (see,Bjornson et al., 283 SCIENCE 534 (1999), incorporated herein byreference).

[0043] In vitro Models of CNS Development, Function and Dysfunction, andMethods for Screening Effects of Drugs on Cells.

[0044] Collagenase-treated neural stem cell cultures cultured in vitrocan be used for the screening of potential neurologically therapeuticcompositions. These compositions can be applied to cells in culture atvarying dosages, and the response of the cells monitored for varioustime periods. Physical characteristics of the cells can be analyzed byobserving cell and neurite growth with microscopy. The induction ofexpression of new or increased levels of proteins such as enzymes,receptors and other cell surface molecules, or of neurotransmitters,amino acids, neuropeptides and biogenic amines can be analyzed with anytechnique known in the art which can identify the alteration of thelevel of such molecules. These techniques include immunohistochemistryusing antibodies against such molecules, or biochemical analysis. Suchbiochemical analysis includes protein assays, enzymatic assays, receptorbinding assays, enzyme-linked immunosorbant assays (ELISA),electrophoretic analysis, analysis with high performance liquidchromatography (HPLC), Western blots, and radioimmune assays (RIA).Nucleic acid analysis such as Northern blots can be used to examine thelevels of mRNA coding for these molecules, or for enzymes whichsynthesize these molecules. Alternatively, cells treated with thesepharmaceutical compositions can be transplanted into an animal, andtheir survival, ability to form neuronal connections, and biochemicaland immunological characteristics examined as previously described.

[0045] The collagenase-treated neural stem cell cultures can be used inmethods of determining the effect of a biological agents on neuralcells. The term “biological agent” refers to any agent, such as a virus,protein, peptide, amino acid, lipid, carbohydrate, nucleic acid,nucleotide, drug, pro-drug or other substance that may have an effect onneural cells whether such effect is harmful, beneficial, or otherwise.Biological agents that are beneficial to neural cells are referred toherein as “neurological agents”, a term which encompasses anybiologically or pharmaceutically active substance that may provepotentially useful for the proliferation, differentiation or functioningof CNS cells or treatment of neurological disease or disorder. Todetermine the effect of a potential biological agent on neural cells, aculture of collagenase-treated neural stem cell cultures is obtained andproliferated in vitro in the presence of a proliferation-inducing growthfactor. Generally, the biological agent will be solubilized and added tothe culture medium at varying concentrations to determine the effect ofthe agent at each dose. The culture medium may be replenished with thebiological agent every couple of days in amounts, so as to keep theconcentration of the agent somewhat constant.

[0046] Thus, it is possible to screen for biological agents thatincrease the proliferative ability of progenitor cells which would beuseful for generating large numbers of cells for transplantationpurposes. It is also possible to screen for biological agents whichinhibit precursor cell proliferation, using collagenase-treated neuralstem cell cultures. Also, the ability of various biological agents toincrease, decrease or modify in some other way the number and nature ofdifferentiated neural cells can be screened on collagenase-treatedneural stem cell cultures that have been induced to differentiate. Theeffects of a biological agent or combination of biological agents on thedifferentiation and survival of differentiated neural cells can then bedetermined. It is also possible to determine the effects of thebiological agents on the differentiation process by applying them tocollagenase-treated neural stem cell cultures prior to differentiation.

[0047] Other features, objects, and advantages of the invention will beapparent from the description and from the claims. In the specificationand the appended claims, the singular forms include plural referentsunless the context clearly dictates otherwise. Unless defined otherwise,all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. All patents and publications cited in thisspecification are incorporated by reference. The following examples arepresented in order to more fully illustrate the preferred embodiments ofthe invention. These examples should in no way be construed as limitingthe scope of the invention, as defined by the appended claims.

EXAMPLE 1 Collagenase Protocol

[0048] 1. Rinse tissue several times with Ca⁺⁺, Mg⁺⁺-free phosphatebuffered saline (PBS).

[0049] 2. In a Petri dish, cut tissue into 1-2 mm cubed pieces usingcrossed scalpels. Pour the tissue (packed volume ˜7.5 ml) and PBS (˜8ml) into a 50 ml centrifuge tube. Rinse the dish with PBS as necessaryto remove tissue, and transfer to the centrifuge tube.

[0050] 3. Centrifuge the tubes gently to sediment the tissue and freecells (<1000 rpm).

[0051] 4. Remove the supernatant carefully using a vacuum line.

[0052] 5. For up to 1.0 g of tissue, add 5.0 ml 0.1% Collagenase, 0.1%Hyaluronidase in 1BSS without Ca⁺⁺, Mg⁺⁺ which contains 1.0% bovineserum albumin (BSA).

[0053] 6. Incubate in 37° C. waterbath with occasional light agitationfor 1 hour. At the end of 1 hr, vortex for approximately 3 sec, andevaluate the extent tissue dissociation. If large pieces of intacttissue remains, continue incubation at 37° C. for another 30-45 min.Stand the tube upright for 1-2 min to allow large cell aggregates tosettle out. Transfer the supernatant to a fresh tube (1).

[0054] 7. Top up the fresh tube (1) with PBS+0.1% BSA and centrifuge atapproximately 900 rpm for 6 min. Remove the supernatant, which should becloudy with debris, to a fresh 50 ml centrifuge tube (2). Recentrifugeboth tubes (1) and (2) at 900 rpm.

[0055] 8. Discard the supernatant from tube (1) and examine the pelletfrom tube (2) for viable cells. If worthwhile, combine the pellettedcells from tube (2) with the contents of tube (1). Fill up the tube withPBS+0.1% BSA and respin for 6 min at 900 rpm, discarding the supernatantwhen finished. Resuspend the cells in PBS and count with trypan blue. Atthis stage, the cell suspension should be relatively free of debris, andshould consist predominantly of healthy cells.

EXAMPLE 2 Collagenase Results

[0056] Collagenase treatment provided increased number of viable neuralstem cells using the collagenase method. Cells are counted in trypanblue on hemocytometer. A raw count is the number of live cells indefined area. TABLE 3 Live Dead Cells/ml Total cells % viabilityCollagenase 121 12 8.07 × 10⁵ 8.07 × 10⁶ 91 Trituration 45 49   3 × 10⁵ 3. × 10⁶ 48

[0057] TABLE 4 Collagenase % Viability % Viability Days TriturationTreatment Trituration Collagenase 0 2.00 × 10⁶ 2.00 × 10⁶ 7 4.60 × 10⁶1.60 × 10⁷ 40.00%   89% 17 1.04 × 10⁷ 7.28 × 10⁷ 52.00%   93% 25 8.80 ×10⁶ 1.67 × 10⁸ 37.00%   78% 33 1.55 × 10⁷ 6.75 × 10⁸ 48.00%   91% 412.33 × 10⁷ 2.61 × 10⁹ 38.00%   98% Average Average 43.00%  90.% ±6.63%±7.4%

[0058] TABLE 5 Input Cell Output Cell % X Accumulated Days TriturationNo. No. Viability Expansion Cell No. 0 P24-P1 2.00 × 10⁶ 2.00 × 10⁶ 7P25-P2 2.00 × 10⁶ 4.60 × 10⁶ 40% 2.3 4.60 × 10⁶ 17 P26-P3 2.00 × 10⁶4.50 × 10⁶ 52% 2.3 1.04 × 10⁷ 25 P27-P4 2.00 × 10⁶ 1.70 × 10⁶ 37% 0.98.80 × 10⁶ 33 P28-P5 1.95 × 10⁶ 3.00 × 10⁶ 48% 1.8 1.55 × 10⁷ 41 P29-P62.00 × 10⁶ 3.00 × 10⁶ 38% 1.5 2.33 × 10⁷ Average 43%

[0059] TABLE 6 Input Cell Accumulated % X Accumulated Days CollagenaseNo. Cell No. Viability Expansion Cell No. 0 P24-P1 2.00 × 10⁶ 2.00 × 10⁶7 P25-P2 2.00 × 10⁶ 1.60 × 10⁷ 89% 8.0 1.60 × 10⁷ 17 P26-P3 2.00 × 10⁶9.70 × 10⁶ 93% 4.6 7.28 × 10⁷ 25 P27-P4 2.00 × 10⁶ 4.60 × 10⁶ 78% 2.31.67 × 10⁸ 33 P28-P5 1.70 × 10⁶ 8.06 × 10⁶ 91% 4.0 7.94 × 10⁸ 41 P29-P62.00 × 10⁶ 5.73 × 10⁶ 98% 3.9 2.27 × 10⁹ Average 90%

[0060] The foregoing description has been presented only for thepurposes of illustration and is not to limit the invention to theprecise form disclosed, but by the claims appended hereto.

We claim:
 1. A method for the in vitro proliferation of a neural stemcell culture comprising the steps of: (a) obtaining dissociated neuraltissue in cell suspension, the suspension containing one or moremultipotent neural stem cells capable of producing progeny that arecapable of differentiating into neurons and glia; (b) culturing the cellsuspension in a culture medium containing at least oneproliferation-inducing growth factor to proliferate the neural stemcells in (a) to generate a neural stem cell culture; and (c) passagingthe cell culture in (b) by treating the culture with an amount of acollagenase preparation effective to dissociate the cells in the cultureand passing the cell culture to a culture medium containing at least oneproliferation-inducing growth factor to further proliferate the neuralstem cell culture.
 2. The method of claim 1 wherein the amount of thecollagenase preparation is between 18-180 mU/ml.
 3. The method of claim1 wherein the amount of the collagenase preparation is between 54-126mU/ml.
 4. The method of claim 1 wherein the collagenase preparationfurther comprises at least one molecule selected from the groupconsisting of sulfhydryl protease, clostripain, aminopeptidase, orcombinations thereof.
 5. The method of claim 1 wherein the collagenasepreparation is substantially pure, and contains minimal secondaryproteolytic activity.
 6. The process of claim 1 , wherein theproliferation-inducing growth factor is selected from the groupconsisting of epidermal growth factor, amphiregulin, acidic fibroblastgrowth factor, basic fibroblast growth factor, transforming growthfactor alpha, leukocyte inhibitory factor (LIF), glycostatin C andcombinations thereof.
 7. The method of claim 1 wherein the neural stemcell culture comprises genetically modified neural stem cells.
 8. Themethod of claim 1 , further comprising the step of differentiating theneural stem cell culture of (c) to produce a cell culture comprisingdifferentiated neural cells selected from the group consisting ofastrocytes, neurons, oligodendrocytes, and combinations thereof.
 9. Themethod of claim 1 or claim 8 , further comprising contacting the neuralstem cell culture with a biological agent, and determining the effectsof the biological agent on cells in the culture.
 10. The method of claim1 wherein the neural stem cell culture is a suspension culture.
 11. Themethod of claim 1 wherein the neural stem cell culture is an adhesionculture.
 12. The method of claim 1 wherein the neural stem cell culturecomprises human neural stem cells.
 13. A method for the in vitroproliferation of a neural stem cell culture comprising the steps of: (a)obtaining dissociated neural tissue in cell suspension, the suspensioncontaining one or more multipotent neural stem cells capable ofproducing progeny that are capable of differentiating into neurons andglia; (b) culturing the cell suspension in a culture medium containingat least one proliferation-inducing growth factor to proliferate theneural stem cells in (a) to generate a neural stem cell culture; and (c)passaging the cell culture in (b) by treating the culture with an amountof a collagenase preparation effective to dissociate the cells in theculture and passing the cell culture to a culture medium containing atleast one proliferation-inducing growth factor to further proliferatethe neural stem cell culture, wherein the percent viability of the cellsin the culture is at least 60% after being passaged.
 14. The method ofclaim 13 , wherein the percent viability of the cells in the culture isat least 75% after being passaged.
 15. The method of claim 13 , whereinthe percent viability of the cells in the culture is at least 85% afterbeing passaged.
 16. The method of claim 13 , wherein the amount of thecollagenase preparation is between 18-180 mU/ml.
 17. The method of claim13 , wherein the amount of the collagenase preparation is between 54-126mU/ml.
 18. The method of claim 13 , wherein the collagenase preparationfurther comprises at least one molecule selected from the groupconsisting of sulfhydryl protease, clostripain, aminopeptidase, orcombinations thereof.
 19. The method of claim 13 , wherein thecollagenase preparation is substantially pure, and contains minimalsecondary proteolytic activity.
 20. The method of claim 13 , wherein theneural stem cell culture comprises genetically modified neural stemcells.
 21. The method of claim 13 , further comprising the step ofdifferentiating the neural stem cell culture of (c) to produce a cellculture comprising differentiated neural cells selected from the groupconsisting of astrocytes, neurons, oligodendrocytes, and combinationsthereof.
 22. The method of claim 13 wherein the neural stem cell cultureis a suspension culture.
 23. The method of claim 13 wherein the neuralstem cell culture is an adhesion culture.
 24. The method of claim 13wherein the neural stem cell culture comprises human neural stem cells.25. A cell culture made according to any one of claims 13-24.
 26. Amethod for expanding a neural stem cell culture comprising the steps of:(a) culturing a neural stem cell culture that contains one or moremultipotent neural stem cells capable of producing progeny that arecapable of differentiating into neurons and glia in a culture mediumcontaining at least one proliferation-inducing growth factor toproliferate the culture; and (b) passaging the cell culture in (a) bytreating the culture with an amount of a collagenase preparationeffective to dissociate the cells in the culture and passing the cellculture to a culture medium containing at least oneproliferation-inducing growth factor to further proliferate the neuralstem cell culture, wherein there is at least a three-fold increase incell number between weekly passages.
 27. The method of claim 26 whereinthere is at least a five-fold increase in cell number between weeklypassages.
 28. The method of claim 26 wherein the neural stem cellculture is a suspension culture.
 29. The method of claim 26 wherein theneural stem cell culture is an adhesion culture.
 30. The method of claim26 wherein the neural stem cell culture comprises human neural stemcells.
 31. A cell culture made according to any one of claims 26-30. 32.A method for expanding a neural stem cell culture comprising the stepsof: (a) culturing a neural stem cell culture that contains one or moremultipotent neural stem cells capable of producing progeny that arecapable of differentiating into neurons and glia in a culture mediumcontaining at least one proliferation-inducing growth factor toproliferate the culture; and (b) passaging the cell culture in (a) bytreating the culture with an amount of a collagenase preparationeffective to dissociate the cells in the culture and passing the cellculture to a culture medium containing at least oneproliferation-inducing growth factor to further proliferate the neuralstem cell culture, wherein the accumulated cell number can be expandedfrom 10⁶ cells to at least 10⁹ cells in less than 49 days.
 33. Themethod of claim 32 wherein the accumulated cell number can be expandedfrom 10⁶ cells to at least 10⁹ cells in less than 42 days.
 34. Themethod of claim 32 wherein the neural stem cell culture is a suspensionculture.
 35. The method of claim 32 wherein the neural stem cell cultureis an adhesion culture.
 36. The method of claim 32 , wherein the neuralstem cell culture comprises human neural stem cells.
 37. A cell culturemade according to any one of claims 32-36.